Project Summary Pyroptosis is a program of cell death involved in the pathogenesis of leading global causes of mortality. Caspase-1 family proteases initiate pyroptosis by releasing the pore-forming portion of gasdermin D, which inserts into the plasma membrane leading to cell lysis. Cellular factors released during pyroptotic lysis cause local and systemic pathology, but processes regulating gasdermin D pore formation and lysis are not well understood. We recently identified the neurosteroid pregnenolone sulfate as a novel inhibitor of pyroptotic lysis, but its mechanism of action is not yet known. Neurosteroids including pregnenolone sulfate have a lipophilic backbone and interact with plasma membrane proteins and lipids, suggesting the hypothesis that these molecules may disrupt formation of gasdermin D pores. This proposal aims to understand how pregnenolone sulfate prevents pyroptotic lysis and determine whether other steroids share this activity. Our preliminary data demonstrate that pregnenolone sulfate prevents lysis during pyroptosis without affecting upstream activation of inflammasomes or caspase-1. The experiments outlined in this proposal will test the hypothesis that pregnenolone sulfate affects pore formation during pyroptosis and assess gasdermin D cleavage, plasma membrane localization and oligomerization. We will also address the alternative hypotheses that pregnenolone sulfate may regulate cell volume to prevent lysis or potentiate membrane repair processes. Together, the results of these experiments will inform a precise molecular understanding of the mechanism of action and reveal a novel strategy to disrupt pyroptotic lysis. The steroid backbone is shared by a large number of molecules, each with specific biological activities. Our preliminary data suggest that there may be molecular determinants for steroid inhibition of pyroptotic lysis, as a related steroid demonstrates reduced potency. We will test a rationally selected library of structurally distinct steroids for the ability to prevent pyroptotic lysis. These experiments will determine whether endogenous steroids demonstrate potency consistent with potential physiologic relevance. In addition, these results will identify the structure-activity relationship for inhibition of pyroptotic lysis to facilitate development of potent and specific molecules and define a new paradigm for disease intervention.